The goal of this research program is to gain an understanding of the enzymatic generation of singlet oxygen (1O2) and of the resultant pathological damage that this reactive species may induce. The role of 1O2 in the photooxidative damage of membranes is also under investigation. It has been observed that cholesterol, an important component of biological membranes, is readily oxidized in a phospholipid membrane matrix by endogenous 1O2 whereas 1O2 generated exogenously is relatively ineffective. It was concluded that 102 when formed in the phospholipid membrane has a sufficiently long lifetime to effect oxygenation of cholesterol; however, 1O2 generated outside the membrane is quenched by water before appreciable diffusion into the membrane can occur. A hydrophilic substrate which exhibits specific reactivity toward 1O2 has been developed in our laboratories. 9,10-Anthracenedipropanoic acid reacts with 1O2 in aqueous solution to yield an isolable endoperoxide which is stable in the medium. There was no reaction with ground state oxygen, superoxide ion or hydrogen peroxide, oxidizing species likely to be present during enzymatic oxidations. Oxidation of the substrate by hydroxyl radical could only be effected upon exposure to 60Co radiation and the product was distinct from that obtained with 1O2. The rate constant for reaction of this substrate with 1O2 is 1.95 x 10 to the 7th power/M/sec This material will be used to evaluate the level of 1O2 in various enzyme systems.